(1) The distribution and density of vasoactive intestinal polypeptide (VIP) immunoreactive acetylcholinesterase (AChE)-containing nerves around the cerebral arteries was studied by using whole mounts with or without lesioning the sphenopalatine ganglia. Abundant VIP and AChE-containing nerves were observed around the cerebral blood vessels in normal rats. VIP and AChE-staining was also demonstrated in neurons within the sphenopalatine ganglia. Lesions of the sphenopalatine ganglia resulted in a marked reduction of both VIP and AChE activity. In many neurons, coexistence of both VIP and AChE was revealed. These results demonstrate that cholinergic neurons from the sphenopalatine ganglia innervate the cerebral vasculature at the base of the brain, and that VIP and AChE coexists within the same nerve fibers. These results reveal that the major cholinergic innervation to the cerebral blood vessels emanates from the sphenopalatine ganglion and that VIP coexists within these neurons. (2) Colocalization of substance P (SP), corticotropin releasing factor (CRF), and AChE was detected by retrograde tracing and immunocytochemical staining in the nucleus tegmentalis dorsalis lateralis (ntdl) projecting to the medial frontal cortex (MFC), septum, and thalamus of the rat. These results suggest that SP and CRF coexist within a subpopulation of ntdl cholinergic neurons that project to a number of forebrain regions including the MFC. Behavioral studies of the effects of SP, CRF, and the cholinergic agonist, carbachol, employed microinjections into the MFC of rats. SP and CFR did not elicit any behavioral effects when administered alone. Carbachol produced a stereotyped motor behavior, consisting of rapid forepaw treading while in an upright posture, resembling "boxing". SP increased carbachol-induced "boxing". CRF decreased carbachol-induced "boxing". One possible functional significance of the coexistence of SP, CRF, and acetylcholine, in neurons projecting to the medial frontal cortex in rats, appears to be a modulatory potentiation of cholinergic response by SP, and a modulatory inhibition of the cholinergic response by CRF.